Recombinant production, purification, and biochemical characterization of a novel L-lactate dehydrogenase from Bacillus cereus NRC1 and inhibition study of mangiferin.

Lactate dehydrogenase (LDH, EC 1.1.1.27) is one of the vital glycolytic conditions, especially during anaerobic conditions. It is a significant diagnostic, prognostic, and monitoring biomarker parameter. A 950-bp DNA fragment containing the gene (LDH) encoding LDH was amplified from Bacillus cereus NRC1. The deduced amino acid sequence reveals that B. cereus LDH (Bc-LDH) is highly homologous to the LDHs of Bacillus organisms. All LDH enzymes have a significant degree of conservation in their active site and several additional domains with unidentified functions. The gene for LDH, which catalyzes lactate synthesis, was cloned, sequenced (accession number: LC706200.1), and expressed in Escherichia coli BL21 (DE3). In this investigation, Bc-LDH was purified to homogeneity with a specific activity of 22.7 units/mg protein and a molecular weight of 35 kDa. It works optimally at pH 8.0. The purified enzyme was inhibited by FeCl2, CuCl2, ZnCl2, and NiCl, whereas CoCl2 was found to boost the activity of Bc-LDH. The molecular docking of the 3D model of the Bc-LDH structure with a natural inhibitor, mangiferin, demonstrated excellent LDH inhibition, with a free binding energy of -10.2 kcal/mol. Moreover, mangiferin is a potent Bc-LDH inhibitor that inhibits Bc-LDH competitively and has one binding site with a Ki value of 0.075 mM. The LDH-mangiferin interaction exhibits a low RMSF value (>1.5 Å), indicating a stable contact at the residues. This study will pave the way for more studies to improve the understanding of mangiferin, which could be considered an intriguing candidate for creating novel and improved LDH inhibitors.

Neurocognitive Function and Its Related Potentials in Children with Beta Thalassemia Major: An Egyptian Study.

BACKGROUND: Repeated blood transfusions and hemolysis in ß-Thalassemia major children lead to iron overload in various organs, including the brain which may cause neurodegeneration. AIM: To evaluate intelligence quotient in children with ß-thalassemia major and healthy counterparts and to assess risk factors that cause cognitive problems. SUBJECTS AND METHODS: This case-control study was performed on 50 children aged 6-16 years old with ß-thalassemia major as patients group and compared with 50 healthy children as a control group of matched age, sex, and social class. Cognitive functions were evaluated by using the Wechsler Intelligence Scale for Children. Serum ferritin and iron were measured by ELISA. RESULTS: There were significantly lower mean performance and full-scale IQ scores of patients group in comparison with controls, whereas no significant differences between both groups as regards to a verbal IQ score. In thalassemic children, block design, comprehension and arithmetic were negatively correlated with age of disease onset, duration of illness and onset of chelation therapy. Serum iron and ferritin were negatively correlated with similarities and digit span. Serum iron levels were negatively correlated with performance IQ score. CONCLUSION: Children with ß-thalassemia major need to receive more academic attention and cognitive assessment to improve their IQ.

Revealing of a novel xylose-binding site of Geobacillus stearothermophilus xylanase by directed evolution.

Xylan saccharification is a key step in many important biotechnological applications. Xylose is the main product of xylan degradation and is a major xylanase inhibitor in a bioreactor; however, xylose-binding site of xylanase is not discovered yet. Evolving of xylose-tolerant xylanase variants will reduce the cost of xylanases in industry. Glycoside hydrolase family-10 thermostable Geobacillus stearothermophilus xylanase XT6 is non-competitively inhibited by xylose with inhibition constant ki equals to 12.2 mM. In the absence of X-ray crystallography of xylanase-xylose complex, unbiased random mutagenesis of the whole xylanase gene was done by error-prone polymerase chain reaction constructing a huge library. Screening a part of the library revealed xylose-tolerant mutants having three mutations, M116I, L131P and L133V, clustered in the N-terminus of α-helix 3. The best xylose-tolerant mutant showed higher ki and catalytic capability than that of the parent by 3.5- and 3-fold, respectively. In addition, kcat increased 4.5-fold and KM decreased 2-fold. The molecular docking of xylose into xylanase XT6 structure showed that xylose binds into a small pocket between N-terminus of α-helices 3 and 4 and close to the three mutations. Mobility of α-helices 3 and 4, which controls catalysis rate, is restricted by xylose binding and increased by these mutations.